Methods of use of the TACI/TACI-L interaction

ABSTRACT

The invention discloses a novel interaction between a TNF receptor (TACI) and its interacting ligand (TACI-L). Also disclosed are methods of screening candidate molecules to determine potential antagonists and agonists of the TACI/TACI-L interaction. The use of the antagonists and agonists as therapeutics to treat autoimmune diseases, inflammation, and to inhibit graft vs. host rejections is further disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/302,863, filed Apr. 30, 1999, which is incorporated herein byreference.

This invention relates generally to the interaction between thetransmembrane activator and CAML-interactor (TACI) protein and itsligand, TACI ligand (TACI-L), use of the interaction in screening assaysthereof, and related kits.

BACKGROUND OF THE INVENTION

Cellular change is often triggered by the binding of an extrinsicelement, such as a ligand, to the extracellular domain of a cell surfacemembrane receptor protein. This binding can result in cellular changesby activating and controlling intracellular signaling cascades and cellregulatory mechanisms. As such, understanding the initial bindinginteraction between the ligand and its receptor protein can be of greatinterest to the scientific community. A greater understanding of thisinteraction would enable one skilled in the art to modulate theresulting signaling cascade governed by the ligand/receptor interactionby selecting agents for co-stimulation or inhibition of the binding ofthe ligand to its receptor.

The tumor necrosis factor (TNF) receptor family is a class of mammaliansignaling molecules that play an important role in protection againstinfection and immune inflammatory responses such as cellular signaltransduction, stimulation of cells to secrete cytokines, cytotoxic Tcell proliferation, general cellular proliferation, lymph nodeformation, bone formation, and bone degradation. TNF-mediated cellularsignaling often involves a molecular activation cascade, during which areceptor triggers a ligand-receptor mediated signal. Alterations in TNFactivation can have profound effects on a multitude of cellularprocesses, such as the activation or inhibition of cell-specificresponses, cell proliferation, inflammatory reactions, and cell death.

The interactions between TNF ligands and receptors may result inone-directional signaling (the interaction of the TNF receptor/ligandtriggers a signaling cascade in the receptor only) or may result inbi-directional or reverse signaling. In the instances of bi-directionalor reverse signaling, the interaction would not only activate thesignaling cascade of the TNF receptor but would also trigger a signalingcascade in a cell bearing the TNF ligand. (S. Wiley et al., Jour. ofImmun., 3235-39 (1996).) Thus, understanding the interaction between aTNF receptor and ligand may result in therapeutic treatments involvingthe inhibition or enhancement of either one or both of the TNF receptoractivity or TNF ligand activity.

One member of the TNF receptor family is the transmembrane activator andCAML-interactor (TACI), a cell surface protein. The TACI protein hasbeen isolated and is described in WO 98/39361. When activated, TACIstimulates the influx of calcium in lymphocytes and initiates theactivation of a transcription factor through a combination of aCa²⁺-dependent pathway and a Ca²⁺-independent pathway. Functions of TACIinclude controlling the response of lymphocytes to cancer and to foreignantigens in infections, graft rejection, and graft-vs.-host disease(GVHD). Furthermore, activation of lymphocyte signaling allows thepositive selection of functional lymphocytes and negative selectionagainst self-reactive clones. (WO 98/39361 at 15.)

TACI modulated signals are often activated by a extracellularligand/receptor interaction, which then triggers an intracellularprotein/protein interaction. One of the intracellular proteins whichbind with the TACI protein has been identified. TACI interacts with thecalcium-signal modulating cyclophilin ligand (CAML), a proteinassociated with the calcium pathway in lymphocytes. According to WO98/39361, after the binding of the extracellular domain of TACI to anextracellular ligand, the cytoplasmic domain of TACI binds CAML,initiating a Ca²⁺-dependent activation pathway, which includes theactivation of the transcription factors, NF-AT, AP-1 and NF_(k)B, afactor implicated in the actions of other members of the TNF-receptorfamily. The regions for the interaction between TACI and CAML weredefined as the cytoplasmic COOH-terminal 126 amino acids of TACI and theNH₂-terminal 201 amino acids of CAML. CAML's ability to act as asignaling intermediate was verified by the inhibition of TACI-inducedactivation of the transcription factor when blocked by adominant-negative mutant. (Von Bülow, G. et al., Science, Vol. 278, p.138-141 (1997).)

Although this interaction between the cytoplasmic domain of TACI andCAML has been identified, little is known about the extracellular ligandwith which TACI interacts to initiate the intracellular cascades. Giventhe important role TACI plays in signal transduction and given thepotential therapies that may arise from the manipulation of thesignaling cascades, there is a need in the art for the identificationand understanding of the interaction of TACI with its signaling ligand.Further, there is a need for the development of assays and therapeuticmethods using the interaction between TACI and its signaling ligand.

Another TNF protein that has been recently discovered is a ligand thathas been designated Neutrokine α, which is described in WO 98/18921.Identical nucleotide and polypeptide sequences have also been disclosedas “TL5” in EP 0869180A1 and as “63954” in WO 98/27114. As a member ofthe TNF family, Neutrokine α polypeptides were described as useful inthe treatment of tumor and tumor metastasis, infections by bacteria,virus and other parasites, immunodeficiencies, inflammatory disease,lymphadenopathy, autoimmune diseases, and GVHD. Neutrokine α was alsodescribed as useful to mediate cell activation and proliferation.Further, Neutrokine α polypeptides were described as primary mediatorsof immune regulation and inflammatory response. (WO 98/18921 at 11; EP0869180A1 at 3.)

As Neutrokine α polypeptides may inhibit immune cell functions, theligand was described as also having a variety of anti-inflammatoryactivities. (WO 98/18921 at 49.) Specifically, it was said thatNeutrokine cc polypeptides could be used as an anti-neovascularizingagent to treat solid tumors and for other non-cancer indications inwhich blood vessel proliferation is not wanted. (Id.) The polypeptidescould also be employed to enhance host defenses against resistantchronic and acute infections and to inhibit T-cell proliferation by theinhibition of IL-2 biosynthesis. Finally, Neutrokine α polypeptidescould also be used to stimulate wound healing and to treat otherfibrotic disorders. (Id.)

As such activities may be modulated by the Neutrokine α polypeptides,knowledge of how the ligand functions would be of significant interestto the scientific community. WO 98/18921, EP 0869180A1 and WO 98/27114,however, fail to identify specific receptors with which Neutrokine ccpolypeptides bind. Identification of the related TNF receptor wouldallow those skilled in the art to identify antagonists which may then beused in therapies to treat the disorders associated with the Neutrokineα polypeptides. Thus, there is a need to greater understand this TNFligand, identify the receptors with which it interacts, and determinehow the interaction functions.

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art by identifyinga novel interaction between the extracellular domain of TACI and theNeutrokine a polypeptide (hereinafter referred to as TACI ligand(TACI-L)), and uses thereof. Specifically, the invention encompasses theidentification of a novel interaction between TACI (SEQ. ID. NO.: 2) andTACI-L (SEQ. ID. NO.: 4).

The present invention provides a screening method for identifyingmolecules that enhance or inhibit the TACI/TACI-L interaction, or thatprevent or inhibit dissociation of a complex formed by TACI and TACI-L.This screening method involves contacting a mixture of cells whichexpress TACI and cells which express TACI-L with a candidate molecule,measuring cellular responses, and detecting the ability of the candidatemolecule to inhibit or enhance the interaction between TACI and TACI-Lor inhibit the dissociation of the complex formed by TACI and TACI-L.Successful inhibition indicates that the candidate molecule is anantagonist. Increased activation of TACI or TACI-L indicates that thecandidate molecule is an agonist. The candidate molecules are preferablysmall molecules, antibodies or peptides.

In a further aspect of the present invention, a solid phase method maybe used to identify small molecules which inhibit the interactionbetween TACI and TACI-L. Using this method, TACI may be bound and isplaced in a mixture with labeled TACI-L. After contact, the amount ofsignal is measured. Diminished levels of signal indicate that thecandidate molecule inhibited the interaction between TACI and TACI-L.

In a still further aspect, the present invention provides a screeningmethod for identifying molecules which mimic the biological activity ofthe TACI/TACI-L interaction. This screening method involves adding acandidate molecule that binds to TACI or TACI-L to a biological assayand comparing the biological effect of the candidate molecule to thebiological effect of TACI/TACI-L complex.

In yet a further aspect, the invention provides for a therapeutic use ofagonists and antagonists of the TACI/TACI-L complex in the treatment ofdiseases modulated by the complex.

In still a further aspect, the invention provides for the antagonistsand agonists of the TACI/TACI-L complex.

Finally, the invention relates to a kit to aid in the abovedeterminations and uses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ. ID. NO.:1) (FIG. 1 a) and deducedamino acid (SEQ. ID. NO.:2)(FIG. 1 b) sequences of the TACI protein.

FIG. 2 shows the nucleotide (SEQ. ID. NO.:3) (FIG. 2 a) and deducedamino acid (SEQ. ID. NO.:4)(FIG. 2 b) sequences of the TACI-L protein.

FIG. 3 shows the amino acid sequence of a polypeptide (SEQ. ID. NO.:5),in which a CMV leader followed by a leucine zipper motif is fused to theN-terminal region of the amino acid sequence of TACI-L.

FIG. 4 shows the results of a plate binding assay capturing TACI-L inwhich the ligand is diluted 1:2. FIG. 4 a demonstrates the results ofthe assay and shows the complete saturation of the receptor bindingsites. FIG. 4 b, the Scatchard plot corresponding to FIG. 4 a,demonstrates the actual number of sites that were bound. From theseresults, an affinity constant of 1.53×10⁻⁹ can be generated.

FIG. 5 shows the results of a plate binding assay capturing TACI-L inwhich the ligand is diluted 1:5. FIG. 5 a demonstrates the results ofthe assay and shows the complete saturation of the receptor bindingsites. FIG. 5 b, the Scatchard plot corresponding to FIG. 5 a,demonstrates the actual number of sites that were bound. From theseresults, an affinity constant of 2.2×10⁻⁹ can be generated.

FIG. 6 shows the results of a plate binding assay capturing HuTACI/Fc.FIG. 6 a graphs the complete saturation of the receptor binding sites.FIG. 6 b, the Scatchard graph which corresponds to FIG. 6 a,demonstrates the actual number of sites that were bound. The Scatchardplot of FIG. 6 b demonstrates a curvilinear binding, with a low affinityconstant of 5.7×10⁻¹⁰ and a high affinity constant of 1.0×10⁻¹⁰.

DETAILED DESCRIPTION OF THE INVENTION

The terms “TACI” and “TACI protein” are used interchangeably to definethe TNF receptor disclosed by WO 98/39361. TACI comprises anextracellular domain, a transmembrane domain, and a cytoplasmic domain.

“Fragments” of TACI encompass truncated amino acid sequences of the TACIprotein that retain the biological ability to bind to TACI-L. An exampleof such a fragment is the extracellular domain. Such fragments areidentified in WO 98/39361, which is incorporated in this application inits entirety.

“Soluble TACI” includes truncated proteins that lack a functionaltransmembrane domain of the protein but retain the biological activityof binding to TACI-L. The soluble, extracellular domain can be used toinhibit cellular activation.

“Homologous analogs” of TACI include isolated nucleic acids of the TACIprotein that are at least about 75% identical to SEQ.ID.NO.:1 and retainthe biological activity of binding to TACI-L. Also contemplated by theterm are embodiments in which a nucleic acid molecule comprises asequence that is at least 80% identical, at least 90% identical, atleast 95% identical, at least 98% identical, at least 99% identical, orat least 99.9% identical to SEQ. ID. NO.:1 and retain the biologicalactivity of binding to TACI-L. Further included are nucleic acids whichare at least 85% similar, at least 95% similar, or at least 99% similarto nucleic acids that encode the amino acids of the TACI protein, asdescribed in SEQ. ID. NO.:2, and that maintain a binding affinity toTACI-L. Still further included are all substantially homologous analogsand allelic variations.

The percent identity and percent similar may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two nucleic acid molecules can be determined by comparingtheir sequences using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 fornon-identities), and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

The terms “TACI-L” and “TACI ligand” are used interchangeably to definethe member of the TNF ligand family disclosed by WO 98/18921. TACI-L isalso disclosed as “TL5” in EP 0869180A1 and as “63954” in WO 98/27114.The full-length TACI-L comprises an extracellular domain, atransmembrane domain, and a cytoplasmic domain. Although the exactlocation of the extracellular, transmembrane, and cytoplasmic domainsmay differ slightly due to different analytical criteria for identifyingthe functional domains, the range of amino acids 1 to 46 generallyrepresents the intracellular domain; amino acids 47 to 72 represent thetransmembrane domain, and amino acids 73-285, the extracellular domain.

“Fragments” of TACI-L encompass truncated amino acids of the TACI-Lprotein that retain the biological ability to bind to TACI. An exampleof such a fragment is the extracellular domain of TACI-L, which bindsTACI. Another example of a TACI-L fragment is amino acids 123-285 of theextracellular domain of the TACI ligand.

“Soluble TACI-L” includes truncated proteins that lack a functionaltransmembrane domain of the protein but retain the biological activityof binding to TACI. The soluble, extracellular domain can be used toinhibit cellular activation.

“Homologous analogs” of TACI-L include isolated nucleic acids of theTACI-L protein that are at least about 75% identical to SEQ.ID.NO.:3 andretain the biological ability to bind to TACI. Also contemplated by theterm are embodiments in which a nucleic acid molecule comprises asequence that is at least 80% identical, at least 90% identical, atleast 95% identical, at least 98% identical, at least 99% identical, orat least 99.9% identical to SEQ.ID.NO.:3 and retain the biologicalability to bind to TACI. Further included are nucleic acids which are atleast 85% similar, at least 95% similar, or at least 99% similar tonucleic acids that encode the amino acids of TACI-L, as described inSEQ. ID. NO.:4 and that maintain a binding affinity to TACI. Stillfurther included are all substantially homologous analogs and allelicvariations.

Sequences are substantially homologous when at least 50% (preferably60%, more preferably 65%, more preferably 75%, more preferably 85%, andmost preferably 99%) of the nucleotides match over the defined length ofthe DNA sequences. Sequences which are substantially homologous can beidentified by comparing the sequences using software known in the art orby the well-known Southern hybridization experiment. Substantiallyhomologous analogs and allelic variations must maintain the samebiological activity as the protein they are homologous to (e.g. bind tothe same receptor or ligand).

The terms “TACI/TACI-L complex” or “TACI/TACI-L interaction” are usedinterchangeably and refer to the protein unit formed by the bindinginteraction of TACI to TACI-L.

The term “TACI/TACI-L fragment complex” includes the protein unitsformed in which at least one binding partner is either a fragment ofTACI or TACI-L (e.g. the binding interaction of a TACI fragment toTACI-L, TACI to a TACI-L fragment, or a TACI fragment to a TACI-Lfragment) or a homologous analog of TACI or TACI-L. The TACI/TACI-Lfragment complex has the same biological activity, effects, and uses asthe TACI/TACI-L complex, as described below. The term “biologicalactivity” includes the binding of TACI to TACI-L or fragments thereof.

The term “biological effects” includes any cellular changes or effectswhich result from a protein-protein interaction or the interaction of aprotein with an agonist or antagonist. Examples of a biological effectof the TACI/TACI-L complex include an increase or decrease in Ca²⁺ ionsresulting from a protein-protein interaction or the activation of thetranscription factors, NF-AT, AP-1 and NF_(k)B.

The TACI/TACI-L interaction is a protein-protein interaction.Protein-protein interactions can be observed and measured in bindingassays using a variety of detection methodologies that include, but arenot limited to, surface plasmon resonance (Biacore), radioimmune basedassays, and fluorescence polarization binding assays. When performed inthe presence of a test compound, the ability of the test compound tomodulate (e.g. enhance or inhibit) the protein-protein binding affinityis measured. In one embodiment of the instant invention, the bindinginteraction between TACI and TACI-L occurs between the extracellulardomain of the TACI protein and amino acids 123-285 of the extracellulardomain of the TACI ligand.

The discovery of the interaction between TACI and TACI-L is described indetail in Examples 1-3. Briefly, a ligand expression construct wastransfected into cells. The cells were incubated with TACI:Fc, boundwith an antibody of TACI:Fc, and followed by a detecting agent. Asoluble form of TACI-L was used in verifying the interaction and wasproduced by fusing a CMV leader sequence followed by a leucine zippermotif to the polypeptide. Other useful leader sequences include IgKappaand Growth Hormone. PCR was used to amplify the cDNA sequence whichencodes the extracellular domain (amino acids 123-285) of TACI-L byusing the restriction sites of specific oligonucleotides. CMV andleucine zipper sequences can be obtained by methods well known in theart, such as by PCR or by enzymatic digestion of previously clonedsequences. These fragments are ligated and inserted into the appropriateexpression vector. (Smith et al., Cell, Vol. 73, 1349-1360.)

The interaction between TACI and TACI-L was further characterized byplate binding assays, as described in Examples 4 and 5. Plate bindingassays were conducted capturing either the TACI protein or the TACIligand. In each instance, a high affinity constant was obtained,demonstrating the close binding interaction between TACI and TACI-L.

The discovery and understanding of the interaction between theextracellular region of TACI and TACI-L can be used to determinepotential agonists or antagonists and to further develop understandingof which cell types TACI-L acts upon. Assays may utilize the interactionbetween TACI-L and TACI to screen for potential inhibitors (antagonists)or enhancers (agonists) of activity associated with TACI-L molecules andidentify candidate molecules which may serve as therapeutically activeagents that enhance, inhibit or modulate the TACI/TACI-L complex.Potential antagonists to the TACI/TACI-L interaction may include smallmolecules, peptides, and antibodies that bind to and occupy the bindingsite of either TACI or TACI-L, causing them to be unavailable to bind toeach other and therefore preventing normal biological activity. Otherpotential antagonists are antisense molecules which may hybridize tomRNA in vivo and block translation of the mRNA into the TACI-L protein.Potential agonists include small molecules, peptides and antibodieswhich bind to TACI or TACI-L and elicit the same or enhanced biologicaleffects as those caused by the binding of TACI to TACI-L.

Small molecules are usually less than 10K molecular weight and possess anumber of physiochemical and pharmacological properties to enhance cellpenetration, resist degradation and prolong their physiologicalhalf-lives. (Gibbs, J., Pharmaceutical Research in Molecular Oncology,Cell, Vol. 79 (1994).) Antibodies, which include intact molecules aswell as fragments such as Fab and F(ab′)2 fragments, may be used to bindto and inhibit the TACI/TACI-L complex by blocking the commencement ofthe signaling cascade. Such activity by the antibodies could be usefulin the treatment of Acute Respiratory Disease Syndrome (ARDS). (WO98/18921 at 57.) It is preferable that the antibodies are humanized, andmore preferable that the antibodies are human. The antibodies of thepresent invention may be prepared by any of a variety of well-knownmethods.

Antagonists may be employed to inhibit (antagonize) the interactionbetween TACI and TACI-L for therapeutic purposes to treat tumor andtumor metastasis and to combat various autoimmune diseases that may bemodulated by the TACI/TACI-L complex, e.g. multiple sclerosis anddiabetes, as well as other disorders, such as viral infection,rheumatoid arthritis, graft rejection, and IgE-mediated allergicreactions. A further disorder that may be treated by antagonists of theTACI/TACI-L interaction is inflammation mediated by the interaction. Ingeneral, the interaction may be used to study cellular processesassociated with TNF-receptors such as immune regulation, cellproliferation, cell death, and inflammatory responses.

Specific screening methods are known in the art and many are extensivelyincorporated in high throughput test systems so that large numbers oftest compounds can be screened within a short amount of time. The assayscan be performed in a variety of formats, including protein-proteinbinding assays, biochemical screening assays, immunoassays, cell basedassays, etc. These assay formats are well known in the art. Thescreening assays of the present invention are amenable to screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides.

A particular example of an assay for the identification of potentialTACI antagonists is a competitive assay which combines TACI-L and acandidate molecule with TACI under the appropriate conditions for acompetitive assay. Either TACI or TACI-L can be labeled so that thebinding may be measured and the effectiveness of the antagonist judged.The label allows for detection by direct or indirect means. Direct meansinclude, but are not limited to luminescence, radioactivity, optical orelectron density. Indirect means include but are not limited to anenzyme or epitope tag.

By observing the effect that candidate molecules have on TACI/TACI-Lcomplexes in various binding assays, on TACI/TACI-L mediated activity infunctional tests, and in cell based screens, molecules that arepotential therapeutics because they can modulate the TACI/TACI-L-bindinginteraction are identified. Such molecules either mimic the biologicalactivity of the TACI/TACI-L complex, prevent the formation of theTACI/TACI-L complex or inhibit dissociation of the TACI/TACI-L complexalready formed. Molecules preventing the interaction of TACI and TACI-Lmay be useful when enhancement of the immune system is desired.Antagonists of the dissociation of the TACI/TACI-L complex may be usefulas immunosuppressants or antiinflammatory agents.

Molecules which inhibit or prevent the dissociation of the TACI/TACI-Lcomplex can be identified by forming the complex in the absence of acandidate molecule, then adding the candidate molecule to the mixture,and changing the conditions so that, but for the presence of thecandidate molecule, TACI would be released from the complex. Theconcentration of the free or bound TACI could then be measured and thedissociation constant of the complex could be determined and compared toa control.

Another method by which molecules which inhibit the interaction betweenTACI and TACI-L can be identified is the solid phase method, in whichTACI is bound and placed in a medium with labeled TACI-L. After contactwith a candidate molecule, the amount of signal produced by theinteraction between TACI and TACI-L is measured. Diminished levels ofsignal, in comparison to a control, indicate that the candidate moleculeinhibited the interaction between TACI and TACI-L. In a furtherembodiment of this method, TACI-L could be bound and TACI labeled.

Screening assays can further be designed to find molecules that mimicthe biological activity of the TACI/TACI-L complex. Molecules whichmimic the biological activity of the TACI/TACI-L complex may be usefulfor enhancing the interaction. To identify compounds for therapeuticallyactive agents that mimic the biological activity of the TACI/TACI-Lcomplex, it must first be determined whether a candidate molecule bindsto TACI or TACI-L. A binding candidate molecule is added to a biologicalassay to determine its biological effects. The biological effects of thecandidate molecule are then compared to the those of the TACI/TACI-Lcomplex.

Thus, the present invention encompasses methods of screening candidatemolecules for their ability to modulate TACI/TACI-L complexes and theirability to modulate activities mediated by TACI/TACI-L complexes. Byobserving the effect that the candidate molecule has on the knownbinding characteristics of TACI, TACI-L or fragments thereof, compoundsthat inhibit or enhance TACI/TACI-L binding can be identified. Typicalcandidate molecules are small molecules, antibodies, or peptides and maybe part of extensive small molecule libraries developed for use inscreening methods. In this context, the identification of smallmolecules which may interact with the TACI protein or the TACI ligandcan be used to develop drugs that modulate the activation pathway andmay allow physicians to treat distinct immune conditions without thenegative side effects present in current therapies. For such therapeuticuses, the agonists or antagonists of the TACI/TACI-L complex identifiedcan be administered through well-known means, including parenterally(subcutaneous, intramuscular, intravenous, intradermal, etc. injection)and with a suitable carrier. Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation instonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

Generally, the conditions for an assay are conditions under which TACIand TACI-L would normally interact. In other words, for an assay toidentify the inhibitor of the TACI//TACI-L interaction, the conditionswould be such that, but for the candidate molecule, TACI and TACI-Lwould bind.

The following examples are offered by way of illustration, and not byway of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference in theirentirety.

EXAMPLE 1 Generation of TACI-FC

This Example describes a method of generating TACI-Fc. The cDNA sequenceencoding the extracellular domain of TACI (amino acids 2-166) wasamplified by PCR using a sense primer(5′-ataaccggtagtggcctgggccggagcaggcgag-3′) (SEQ. ID. NO. 6) and anantisense primer (5′-ataagatctgggctcgctgtagaccagggccacctgatc) (SEQ. ID.NO. 7). The amplified PCR fragment was digested with the appropriaterestriction enzyme and then ligated into the mammalian expression vectorpDC409, in-frame with the Ig kappa leader sequence at the 5′ end andwith the Fc portion of human IgG1 at the 3′ end. The plasmid wastransfected transiently in CV1/EBNA cells and the soluble proteinTACI-FC was purified on a protein G-sepharose column. Proteinconcentration was determined by BCA analysis. Purity was assessed bySDS-PAGE analysis which, under reducing conditions, showed a single bandat 42 kDa.

EXAMPLE 2 Ligand Screening by Slide Binding Assay

This Example describes the method of a slide binding assay anddemonstrates that the TACI-Fc protein interacted only with TACI-L. Thepurified TACI-Fc was used to screen against a cDNA panel containingknown members of the ligand family (4-1BBL, CD40L, OX40L, CD27L, CD30L,RANKL, LT-alpha, LT-beta, LIGHT, TWEAK, FasL, TRAIL, proTNF and TACI-L).TACI-Fc was then bound to the slides by adding 2 μg of the DNA encodingthe members of the ligand family to a sterile tube and adding 75 μMcholoroquine in transfection/growth medium to a final volume of 175 μl.25 μl of DEAE-dextran (4 mg/ml in PBS) was then added to the DNAsolution and mixed.

The growth medium was aspirated from the slides and replaced with 3 mlof 75 μM chloroquine in the transfection/growth medium, followed by theaddition of the DNA/DEAE-dextran mixture to the cells. The slides wererocked side-to-side and back-and-forth to distribute the precipitatedDNA evenly. The slides were incubated at 37° C. for 4.5 hours.

The medium was aspirated and 3 ml 10% DMSO was added in thetransfection/growth medium. After a 5 minute incubation period at roomtemperature, the medium was aspirated again and replaced with 3 ml freshtransfection/growth medium. The cells were then incubated at 37° C. for2 days to allow for expression of the transfected cDNAs.

To screen for positive pools expressing the cell-bound protein, slideswere incubated with TACI:Fc and then with a radioiodinated protein probe(labeled goat anti-human Fc F(ab′)2) for 30 minutes at room temperature.The probe solution is then removed by aspiration and washed to removethe non-specifically bound probe. Finally, the slides were fixed byincubating each slide with 1 ml 2.5% glutaraldehyde in PBS for 30minutes at room temperature to retain specifically bound label. Theslides were then washed twice with 1 ml PBS and air-dried.

The dried slides were dipped in liquid photographic emulsion that hasbeen warmed to 42° C., dried at room temperature and exposed for 2 daysat room temperature before developing. The slides were examined at 25×magnification under bright-field illumination to detect cell types uponwhich the ligand is acting. TACI-Fc protein was found to bind only tocells transfected with the TACI-L. The ability of the TACI-FC to bind toCV1 expressing the TACI-L was also demonstrated by the well-knownmethods of flow cytometry.

EXAMPLE 3 Immunoprecipitation of Membrane—Associated TACI with theTACI-Ligand

This Example demonstrates the interaction between the TACI protein andTACI-L. CV1 cells were transfected with soluble TACI-L plasmid and thetwo day supernatant was harvested. CV1 cells were transfected withmembrane associated TACI and metabolically labeled with 35S-CYS-MET twodays post-transfection (labeled cell lysate). Supernatant containingTACI-L was used in immunoprecipitation experiments with labeled celllysate. A specific band at 45 kDa which was consistent with thepredicted size of TACI was obtained, as shown in FIG. 1. Thus, theinteraction between the TACI protein and TACI-L was confirmed.

EXAMPLE 4 Plate Binding Assay Capturing TACI-L

This example further characterizes the interaction between TACI andTACI-L by conducting a plate binding assay and demonstrates the highaffinity between the proteins. Equilibrium binding isotherms weredetermined in 96-well microtiter plates that had been coated with TACI-LCOS expressed supernatants, captured through Leucine Zipper M15antibody. Plates were incubated with 5 μg/ml LZ M15 in PBS for 4 hr at4° C. After being washed 3 times with PBS, the plates were incubatedwith a 1:2 or 1:5 dilution of the COS expressing TACI supernatant inPBS/0.05% Tween 20 for 12 hours at 4° C. The plates were then washed foran additional 3 times with PBS and nonspecific binding sites wereblocked with 300 μl/well of a binding media (RPMI 1640, 2.5% BSA, 20mMHEPES, 0.02% sodium azide pH 7.2) and 2.5% non-fat dried milk. Theplates were incubated for 1 hour at room temperature and washed 3 timeswith PBS.

HuTACI/Fc was diluted to 2 μg/ml to the first well, and serial dilutionswere performed against the binding media. Incubation occurred for 2hours at 4° C. Plates were then washed 3 times with PBS. A finalincubation occurred for 30 minutes at room temperature with 125 ng/ml125-I goat anti-human F(ab′)2. The goat anti-human F(ab′)2 was labeledwith 125-I using solid phase chloramine T analog (Iodogen; PierceChemical, Rockford, Ill.) to a specific radioactivity of 8.73e14cpm/mmol. Nonspecific binding was determined in the presence of1000-fold excess of unlabeled goat anti-human F(ab′)2. Plates werewashed 3 times in PBS and the specifically bound ligand was releasedwith 50 mM citrate (pH 3.0) and then gamma counted. Data was processedas described (Dower et al., 1984).

FIG. 4 a demonstrates the results of the assay using 1:2 dilution andshows the complete saturation of the receptor binding sites. FIG. 4 b,the Scatchard graph corresponding to FIG. 4 a, demonstrates the actualnumber of sites that were actually bound. From these results, theaffinity constant of 1.53×10⁻⁹ can be generated.

FIG. 5 a demonstrates the results of the assay using 1:5 dilution andshows the complete saturation of the receptor binding sites. FIG. 5 b,the Scatchard graph corresponding to FIG. 5 a, demonstrates the actualnumber of sites that were actually bound. From these results, theaffinity constant of 2.2×10⁻⁹ is shown.

EXAMPLE 5 Plate Binding Assay Capturing HuTACI/FC

This example also characterizes the interaction between TACI and TACI-Lby use of a plate binding assay and further demonstrates the highaffinity between the proteins. Equilibrium binding isotherms weredetermined in 96-well microtiter plates that had been coated withHuTACI/Fc, captured through goat anti-human Fc polyclonal antibody.Plates were incubated with 5 μg/ml goat anti-human FC in PBS for 4 hoursat 4° C. After being washed 3 times with PBS, the plates were incubatedwith 0.1 μg/ml Fc chimera in PBS/0.05% Tween 20 for 12 hours at 4° C.and then washed for an additional 3 times with PBS. Nonspecific bindingsites were blocked with 300 μl/well of a binding media (RPMI 1640, 2.5%BSA, 20MM HEPES, 0.02% sodium azide pH 7.2) and 2.5% non-fat dried milk.The plates were incubated for 1 hour at room temperature and then washed3 times with PBS. TACI-L was expressed in COS cells and concentrated10-fold.

TACI-L supernatant was diluted 1:10 to the first well, and serialdilutions were performed against the binding media. Incubation occurredfor 2 hours at 4° C. Plates were then washed 3 times with PBS. A finalincubation occurred for 30 minutes at room temperature with 125-ILeucine Zipper M15. Leucine Zipper M15 (LZM15) was labeled with 125-Iusing solid phase chloramine T analog (Iodogen; Pierce Chemical,Rockford, Ill.) to a specific radioactivity of 8.73e14 cpm/mmol.Nonspecific binding was determined in the presence of 1000-fold excessof unlabeled LZM15. Plates were washed 3 times in PBS and specificallybound ligand was released with 50 mM citrate (pH 3.0) and then gammacounted. Data was processed as described (Dower et al., 1984).

FIG. 6 a demonstrates the complete saturation of the receptor bindingsites. FIG. 6 b, the Scatchard graph which corresponds to FIG. 6 a,demonstrates the actual number of sites that were actually bound. TheScatchard graph of FIG. 6 b demonstrates a curvilinear binding, with alow affinity constant of 5.7×10⁻¹⁰ and a high affinity constant of1.0×10⁻¹⁰. FIG. 6 b demonstrates that the majority of the bindingoccurred at an affinity constant between 2−3×10⁻⁹.

1. A method of screening a test compound comprising the steps of: a.forming a composition comprising (i) a first isolated protein,comprising a polypeptide selected from the group consisting of: (a) thepolypeptide of SEQ ID NO:2; (b) a polypeptide comprising amino acids2-166 of SEQ ID NO:2; (c) a fragment of the polypeptide of SEQ ID NO:2;or (d) a polypeptide encoded by a nucleic acid sequence that is at least95% identical to SEQ ID NO: 1; wherein said fragment of (i)(c) and saidpolypeptides of (i)(d) bind SEQ ID NO:4; (ii) a second isolated protein,comprising a polypeptide selected from the group consisting of: (a) thepolypeptide of SEQ ID NO:4; (b) a polypeptide comprising amino acids123-285 of SEQ ID NO:4; or (c) a polypeptide comprising amino acids73-285 of SEQ ID NO:4; (iii) a test compound; and b. assaying for thelevel of interaction of the protein of (i) and the protein of (ii),wherein the affinity constant for protein (i) and protein (ii) is from1.53×10⁻⁹ to 2.2×10⁻⁹; such that if the level obtained in step (b)differs from that obtained in the absence of the test compound, a testcompound that affects the interaction of the protein of (i) and theprotein of (ii) is identified.
 2. The method of claim 1 wherein at leastone of the proteins of (i) and the proteins of (ii) is labeled with adetectable moiety.
 3. The method of claim 1 wherein both the proteins of(i) and (ii) are soluble.
 4. The method of claim 3 wherein both thesoluble protein of (i) and the soluble protein of (ii) are labeled witha detectable moiety.
 5. The method of claim 1 wherein the test compoundis an antibody.
 6. The method of claim 5 wherein the antibody is ahumanized antibody.
 7. The method of claim 1 wherein the composition isformed by adding the test compound to the protein of (i) and the proteinof (ii).
 8. The method of claim 1 wherein step (b) comprises determininga dissociation constant of the interaction of the protein of (i) withthe protein of (ii).
 9. The method of claim 1 wherein step (b) comprisesassessing activation of the protein of (i) in a cell.
 10. The method ofclaim 9 wherein assessing activation of the protein of (i) in a cell ismeasured by calcium influx.
 11. The method of claim 1 wherein theprotein of (ii) is a polypeptide comprising amino acids 123-285 of SEQID NO:4 or a polypeptide comprising amino acids 73-285 of SEQ ID NO:4.12. The method of claim 11 wherein the polypeptide comprising aminoacids 123-285 of SEQ ID NO:4 or the polypeptide comprising amino acids73-285 of SEQ ID NO:4 further comprises a leucine zipper domain.
 13. Themethod of claim 1 wherein the protein of (i) is a polypeptide comprisingamino acids 2-166 of SEQ ID NO:2.
 14. The method of claim 13 wherein thepolypeptide comprising amino acids 2-166 of SEQ ID NO:2 furthercomprises a Fc domain.
 15. A method of screening a test compoundcomprising the steps of: a. forming a composition comprising (i) anisolated protein selected from the group consisting of: (a) thepolypeptide of SEQ ID NO:2; (b) a polypeptide comprising amino acids2-166 of SEQ ID NO:2; and (c) a fragment of the polypeptide of SEQ IDNO:2; wherein said fragment binds SEQ ID NO:4; (ii) the polypeptide ofSEQ ID NO:4; and (iii) a test compound; and b. assaying for the level ofinteraction of the protein if (i) and the protein of (ii), wherein theaffinity constant for protein (i) and protein (ii) is from 1.53×10⁻⁹ to2.2×10⁻⁹; such that if the level obtained in step (b) differs from thatobtained in the absence of the test compound, a test compound thataffects the interaction of the protein of (i) and the protein of (ii) isidentified.
 16. A method of screening a test compound comprising thesteps of: a. forming a composition comprising (i) the polypeptide of SEQID NO:2; (ii) an isolated protein selected from the group consisting of:(a) the polypeptide of SEQ ID NO:4; (b) a polypeptide comprising aminoacids 123-285 of SEQ ID NO:4; (c) a polypeptide comprising amino acids73-285 of SEQ ID NO:4; and (d) a fragment of the polypeptide of SEQ IDNO:4; wherein said fragment binds SEQ ID NO:2; and (iii) a testcompound; and b. assaying for the level of interaction of the protein of(i) and the protein of (ii), wherein the affinity constant for protein(i) and protein (ii) is from 1.53×10⁻⁹ to 2.2×10⁻⁹; such that if thelevel obtained in step (b) differs from that obtained in the absence ofthe test compound, a test compound that affects the interaction of theprotein of (i) and the protein of (ii) is identified.
 17. A method ofscreening a test compound comprising the steps of: a. forming acomposition comprising (i) the polypeptide of SEQ ID NO:2; (ii) thepolypeptide of SEQ ID NO:4; and (iii) a test compound; and b. assayingfor the level of interaction of the polypeptide of (i) and thepolypeptide of (ii), wherein the affinity constant for protein (i) andprotein (ii) is from 1.53×10⁻⁹ to 2.2×10⁻⁹; such that if the levelobtained in step (b) differs from that obtained in the absence of thetest compound, a test compound that affects the interaction of theprotein of (i) and the protein of (ii) is identified.
 18. The method ofclaim 11, wherein the polypeptide comprising amino acids 123-285 of SEQID NO:4 or the polypeptide comprising amino acids 73-285 of SEQ ID NO:4further comprises a Fc domain.
 19. The method of claim 5, wherein theantibody is human.
 20. The method of claim 5, wherein the antibodycomprises a Fab fragment.
 21. The method of claim 5, wherein theantibody comprises a F(ab′)₂ fragment.
 22. A method of screening a testcompound comprising the steps of: a. forming a composition comprising(i) a first isolated protein, comprising a polypeptide selected from thegroup consisting of: (a) the polypeptide of SEQ ID NO:2; or (b) apolypeptide comprising amino acids 2-166 of SEQ ID NO:2; (ii) a secondisolated protein, comprising a polypeptide selected from the groupconsisting of: (a) the polypeptide of SEQ ID NO:4; (b) a polypeptidecomprising amino acids 123-285 of SEQ ID NO:4; (c) a polypeptidecomprising amino acids 73-285 of SEQ ID NO:4; (d) a fragment of thepolypeptide of SEQ ID NO:4; or (e) a polypeptide encoded by a nucleicacid sequence that is at least 95% identical to SEQ ID NO:3; whereinsaid fragment of (ii)(d) and said polypeptides of (ii)(e) bind SEQ IDNO:2; and (iii) a test compound; and b. assaying for the level ofinteraction of the protein of (i) and the protein of (ii), wherein theaffinity constant for protein (i) and protein (ii) is from 1.53×10⁻⁹ to2.2×10⁻⁹; such that if the level obtained in step (b) differs from thatobtained in the absence of the test compound, a test compound thataffects the interaction of the protein of (i) and the protein of (ii) isidentified.